Vacuum manifold for filtration microscopy

ABSTRACT

A vacuum manifold for filtration microscopy includes a manifold top having multiple openings, and a capture membrane positioned above and spaced apart from the manifold top, where the capture membrane is configured to deflect into contact with a surface of the manifold top when a negative pressure is applied to the multiple openings. A method for filtration microscopy includes the steps of providing a vacuum manifold including a manifold top having a plurality of openings, and a capture membrane positioned above and spaced apart from the manifold top; applying sample drops to sample spots on the membrane, the sample spots positioned above the plurality of openings; applying a negative pressure to the openings such that the capture membrane contacts a surface of the manifold top; and optically imaging particulates on the capture membrane.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. non-provisional applicationSer. No. 16/394,173, filed on Apr. 25, 2019, which claims priority toU.S. provisional application No. 62/662,370, filed on Apr. 25, 2018,incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Particle analysis is a growing market that impacts most industrialsectors spanning from healthcare to cements. Particles can span a widesize range from a few nanometers up to several centimeters and need tobe analyzed for research, process and quality control purposes.

Optical microscopy is a key technique for conducting particle analysis,offering particle sizing, counting, morphology and visualization basedon image analysis. Depending on the sample's optical properties and themicroscope's resolution, optical microscopy can be used to analyzeparticles ranging from a few hundred nanometers up to centimeter scale.Optical microscopy enables key advantages over other particle analysismethods, particularly ensemble-based methods like dynamic or staticlight scattering, since it offers individual particle analysis andextracting additional information including morphology and potentiallyoptical and spectroscopic properties of the particles.

One modification of optical microscopy used to handle wet dispersions isfiltration microscopy, also known as membrane microscopy. In membranemicroscopy, a sample containing particles suspended in a solution isfiltered through a porous membrane, and particles larger than the poresizes remain on top of the filter (unfiltered) and are later imaged andanalyzed. Membrane microscopy is typically used to assess processstability and screen for impurities. Membrane imaging is used in severalindustries with applications including but not limited to:

Pharmaceuticals: Filtration microscopy, also known membrane microscopyis the original quality control method for measuring subvisibleparticles (1 um-100 um) in pharmaceutical injectables. A non-automatedversion of this microscopy method became the first USP method for lotrelease (USP 788) recommending the acceptable levels of particulatecontent in pharmaceutical injectables, including but not limited tosmall molecule dispersions and biopharmaceuticals. This is a veryimportant step since the presence of undesired particles inpharmaceutical solutions may be correlated to immunogenicity. Filtrationmicroscopy can also be used to characterize adjuvants in vaccines andpharmaceuticals particles used for encapsulation or drug delivery.

Oil and Gas: Membrane microscopy is applied in several applicationsincluding analyzing micrometer scale fracking agents and muds tomeasuring undesired sediment in jet fuels batches.

Aquatic and water quality research: Filtration microscopy can be used tomeasure algae, plankton and other bacteria and undesired pathogens inwater sources.

Chemicals: Filtration microscopy can be used to measure nano ormicrometer scaled precipitated chemicals and impurities present in thesebottles.

Food and Beverage: Microparticles can be used in encapsulation andcharacterized via membrane microscopy.

Yet, optical microscopy faces two key challenges for conducting properanalysis on wet dispersions. The first challenge is not confounding theporous membrane texture with the particles being measured. The secondchallenge is ensuring that the particles are sufficiently dispersed onthe membrane such that there is little to no particle stacking so thatindividual particles can be resolved and analyzed.

Thus, there is a need in the art for a device, system and method thatcan perform more reliable and accurate optical microscopy analysis onwet dispersions while minimizing the issues with confounding the porousmembrane texture with the particles being measured and particlestacking.

SUMMARY OF THE INVENTION

In one embodiment, a vacuum manifold for filtration microscopy includesa manifold top including multiple openings; and a capture membranepositioned above and spaced apart from the manifold top, where thecapture membrane is configured to deflect into contact with a surface ofthe manifold top when a negative pressure is applied to the multipleopenings. In one embodiment, multiple opening are each defined by acontacting surface protruding from the manifold top. In one embodiment,the contacting surface protruding from the manifold top is a contactingring. In one embodiment, the contacting surface protruding from themanifold top includes one or more pins. In one embodiment, the capturemembrane further includes hydrophobic rings defining the sample spots.In one embodiment, the hydrophobic rings are positioned directly abovethe contacting rings. In one embodiment, at least a portion of thecontacting rings are positioned directly below sample spots defined bythe hydrophobic rings. In one embodiment, the vacuum manifold includes ahousing defining a cavity and configured to seat the manifold top, wherethe cavity is in fluid communication with the multiple openings. In oneembodiment, the vacuum manifold includes a vacuum port in fluidcommunication with the cavity and configured to connect to a vacuumsupply. In one embodiment, the multiple openings are 96 openings. In oneembodiment, the vacuum manifold includes a drying cassette configured toseat blotting paper and the capture membrane. In one embodiment, thecapture membrane comprises pore sizes between about 50 nanometers and 10microns. In one embodiment, the vacuum manifold is in ANSI 96 well sizeformat. In one embodiment, the capture membrane is a 25 mm or a 47 mmmembrane filter. In one embodiment, at least a portion of a surface ofthe manifold top or capture membrane includes a non-stick coating. Inone embodiment, the vacuum manifold includes a pressure control openingconfigured in the manifold top and separate from the multiple openings.In one embodiment, a space defined by two or more of the rings, thecapture membrane and the manifold top is configured to generate apressure higher than a negative pressure applied to the multipleopenings and lower than atmospheric pressure.

In one embodiment, a method for filtration microscopy includes the stepsof providing a vacuum manifold including a manifold top having multipleopenings, and a capture membrane positioned above and spaced apart fromthe manifold top; applying sample drops to sample spots on the membrane,the sample spots positioned above the openings; applying a negativepressure to the openings such that the capture membrane contacts asurface of the manifold top and fluid from the plurality of sample dropsis suctioned through the capture membrane; and optically imagingparticulates on the capture membrane. In one embodiment, the pluralityof sample drops includes a biopharmaceutical solution. In oneembodiment, the biopharmaceutical solution includes visible particles,subvisible particles and nanometer scale particles. In one embodiment,the particulates include protein aggregates. In one embodiment, theparticulates include at least one of polysorbate particles, metallicparticles, rubber particles, silicone oil droplets, salt crystals,cellulose and sucrose particles. In one embodiment, the sample dropsinclude a viscosity between 1 cP and 200 cP. In one embodiment, thesample drops include a tag to perform at least one of fluorescentidentification and protein activity monitoring. In one embodiment, themethod includes the step of applying a drop comprising a tag after thestep of applying a negative pressure to perform at least one offluorescent identification and protein activity monitoring. In oneembodiment, the method includes the step of generating a datavisualization image based on the imaging and indicative of dispersion ofthe particulates. In one embodiment, the capture membrane furtherincludes multiple hydrophobic rings defining the multiple sample spots,and the step of optically imaging includes matching an imaging devicefield of view to the size and shape of a single hydrophobic ring forwhole well imaging.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing purposes and features, as well as other purposes andfeatures, will become apparent with reference to the description andaccompanying figures below, which are included to provide anunderstanding of the invention and constitute a part of thespecification, in which like numerals represent like elements, and inwhich:

FIGS. 1A and 1B are diagrams of a sample (pre-vacuum and during vacuumrespectively) containing particulates on a capture membrane according toone embodiment.

FIGS. 2A and 2B are diagrams of a cross-sectional view of a manifold topand capture membrane (pre-vacuum and during vacuum respectively)according to one embodiment.

FIGS. 3A-3B are images of vacuum manifold assemblies according tovarious embodiments. FIG. 3A is a perspective view of an assembledvacuum manifold, without the drying cassette; FIG. 3B is a perspectiveview of unassembled vacuum manifold components; FIG. 3C is a perspectiveview of a vacuum of an assembled vacuum manifold without the capturemembrane; FIG. 3D is a magnified view of a manifold top; FIG. 3E is aperspective view of an assembled vacuum manifold; and FIG. 3F is aperspective view of a vacuum manifold drying cassette and blottingpaper.

FIG. 4 is an image of a data visualization tool according to oneembodiment.

FIG. 5 is a flow chart of a method for filtration microscopy accordingto one embodiment.

FIGS. 6A-6J illustrate a laminar flow fluid mechanics simulationprogression and a magnified view 6K according to one embodiment.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that the figures and descriptions of the presentinvention have been simplified to illustrate elements that are relevantfor a clearer comprehension of the present invention, while eliminating,for the purpose of clarity, many other elements found in systems andmethods of optical microscopy. Those of ordinary skill in the art mayrecognize that other elements and/or steps are desirable and/or requiredin implementing the present invention. However, because such elementsand steps are well known in the art, and because they do not facilitatea better understanding of the present invention, a discussion of suchelements and steps is not provided herein. The disclosure herein isdirected to all such variations and modifications to such elements andmethods known to those skilled in the art.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

As used herein, each of the following terms has the meaning associatedwith it in this section.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20%, ±10%, ±5%, ±1%, and ±0.1% from the specified value,as such variations are appropriate.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Where appropriate, the description of a range should beconsidered to have specifically disclosed all the possible subranges aswell as individual numerical values within that range. For example,description of a range such as from 1 to 6 should be considered to havespecifically disclosed subranges such as from 1 to 3, from 1 to 4, from1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well asindividual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5,5.3, and 6. This applies regardless of the breadth of the range.

Referring now in detail to the drawings, in which like referencenumerals indicate like parts or elements throughout the several views,in various embodiments, presented herein is a device, system and methodfor filtration microscopy.

Embodiments described herein include a filtration manifold assembly forfiltration microscopy that ensures laminar flow to reduce particlestacking and produce better quality particle analysis, further removingthe undesired post filtrate at the bottom of the membrane to improveanalysis. The vacuum manifold is an assembly that enables highly uniformspatial distribution of particles present in liquid dispersions onto acapture membrane for improved particle analysis, as illustrated in FIGS.1A and 1B. In order to get accurate particle sizes and counts, it isadvantageous that particles from the liquid sample are deposited on thecapture membrane in such a way as to minimize particle overlap.Particles that are touching or overlapping with each other could becounted as one, larger particle, rather than two smaller ones. There aretwo important considerations. First, it's best to maximize the givenmembrane area available so that particles are less likely to be randomlyplaced in proximity with each other. It would be less optimal to guidethe flow of liquid through a small region of the available membrane sothat all of the particles are concentrated into this smaller region.This would increase the probability of particle overlap. Second, theflow rate of the liquid through the membrane will aid the separation ofthe particles. If a solution containing particles were to be placed onthe membrane and allowed to sit for a long period of time, particlesthat are less dense than the liquid will sediment onto the membrane.Their positions on the membrane will be random. Instead, if the fluid isdriven through the membrane with a pressure differential and allowed toflow through the membrane, particles that have already landed on themembrane will block the membrane pores underneath them. In theseconditions of laminar flow, the fluid will flow around these sedimentedparticles, directing other particles away from them. In this way, theparticles are less likely to touch each other.

With reference now to FIGS. 2A and 2B, a cross-sectional view of vacuummanifold 100 illustrating a manifold top 114 and capture membrane 106 isshown before (FIG. 2A) and during (FIG. 2B) the application of vacuumaccording to one embodiment. The capture membrane 106 has sample spots104 defined by hydrophobic rings 102. A sample drop 108 is shown aswell, contained within the hydrophobic ring 102 that defines a samplespot 104. During vacuuming, the sample drop 108 is pulled down throughthe membrane 106 and then through the holes of the manifold top 114 andinto the manifold cavity 130. In one embodiment, there are 96 samplespots 104. Alternate embodiments may include other standardconfigurations, for example 96, 24, 12 and 6 sample spots. Each of thesample spot 104 aligns with an opening 124 in the manifold top 114.Three distinct pressure zones are present, including zone 1 or highpressure (atmosphere) 110, zone 2 or low pressure 112 directly under thesample spot 104, and zone 3 or medium pressure 116 between contactingrings 126. The geometry of the manifold top 114 includes contactingrings 126 that protrude from the top of the manifold top to align withthe hydrophobic rings 102. A pressure control hole (see e.g. FIG. 3D)which in one embodiment has an adjustable aperture can be included tofine-tune pressure levels. The pressure control hole controls thepressure in the medium pressure zone which also aids in reducing noisethat otherwise occurs during vacuum.

The contacting rings 126 contact the membrane 106 within the samplesspot 104 area and contact region 121 when vacuum is applied. Thiscontact disrupts the surface tension, allowing the sample drop 108 toflow through the membrane 106. The contact region 121 in one embodimentcan be along the perimeter of the sample spot 104 area. The design ofthe manifold top 114 is such that at rest (FIG. 2A), the sample spot 104of the membrane 106 does not touch the contacting rings 126 beneath themembrane 106, since there is a gap 120. However, the ring 126 is closeenough to the membrane 106 so that that when a threshold vacuum pressureis applied (FIG. 2B), the deflection of the membrane 106 initiatescontact 120′ between the membrane 106 and the contacting rings 126 atthe contact region 121. This contact 120′ between the membrane 106 andthe contacting rings 126 initiates flow through the membrane 106 bybreaking surface tension capillary forces. In one embodiment, an edge122 of the contacting ring is angled or curves down into the hole 124 tofacilitate initiation of flow through the membrane 106 and into the hole124. In one embodiment, the gap 120 is about 0.1 mm. Generally, the gap120 should be far enough away so that the weight of the liquid doesn'tdeflect the membrane 106 and initiate contact with the contacting rings126 before the threshold vacuum pressure is reached. Otherwise, if thegap 120 is too big, the membrane 106 will be too far away to contact thecontacting rings 126 during vacuuming or the membrane 106 may bestretched too far and will become deformed. In one embodiment, between10 and 50 kPa of vacuum pressure is applied during vacuum cycles.

In one embodiment, each of the 96 sample spots 104 on the capturemembrane 106 is above a hole in the manifold top 114. Each of the 96holes allows the vacuum pressure within the manifold cavity 130 to drawliquid through the corresponding sample spot 104 above the hole 124.Importantly, the contacting ring 126 surrounds each hole 124 in themanifold top 114 and contacts the sample spot 104 region of the capturemembrane 106 from the bottom. Contacting the top surface of the contactring 126 with the sample spot 104 is what breaks the surface tensionthat otherwise opposes flow of the sample drop 108 through the capturemembrane 106. In one embodiment, there are 96 contacting rings 126 thatmatch geometrically with the 96 sample spots 104 on the capture membrane106. With reference now to the vacuum manifold assembly 200 shown inFIG. 3A, in one embodiment, the vacuum manifold assembly 200 includes anexternal housing 280 that seats a manifold top 214. The manifold top 214seats a capture membrane 206 which in one embodiment has 96 hydrophobicrings 202 defining 96 sample spots 204 for containing sample drops 208.A vacuum source 250 connects to a cavity below the membrane 206 via avacuum connection port 262 and tubing 252. With reference now to FIG.3B, blotting paper 268, drying cassette 266, manifold top 214 andmanifold bottom 260 components are shown. The manifold bottom 260includes a cavity 230 connected to and in fluid communication with avacuum connection port 262, for connecting to tubing and a vacuumsource. As shown in FIG. 3C, the manifold top 214 and bottom 260 areassembled and ready to accept a capture membrane for sample processing.As shown in FIG. 3D, a pressure hole 270 can be positioned in themanifold top 214 to regulate system pressure. In one embodiment, thepressure hole 270 can have an adjustable opening that opens and closesto adjust internal system pressure, and can also be automated based on apressure feedback measurement. Contacting rings 202 are also shownprotruding from the manifold top 214. As shown in FIG. 3E, the manifoldtop 214 and bottom 260 are assembled with a capture membrane 206 placedin the pocket of the manifold top 214. With reference to FIG. 3F, themanifold top 214, bottom 260, drying cassette and blotting paper 268 areassembled. This configuration is ready to accept a capture membrane fordrying the plate bottom. In one embodiment, membranes can be configuredto process from 1 ul of sample to 100 ml of sample.

Embodiments of the vacuum manifold can be customized or adjusted fordifferent filter sizes, shapes and characteristics. For example, in oneembodiment, the vacuum manifold is a 96 well filter plate in ANSIformat. In one embodiment, the vacuum manifold is a circular shapedfilter, 5 mm to 10 cm in diameter. In one embodiment, the pore sizes inthe membrane are between 20 nm and 10 um. In one embodiment, the poresizes in the membrane are between 50 nm and 10 um. In one embodiment,the pore sizes in the membrane are about 440 nm. In one embodiment,different membrane materials and coatings are utilized, such as, pvp andpeg, chrome, gold. In one embodiment, the membrane material is apolymeric, ceramic or metallic material. In one embodiment, a polymericmembrane material is a polycarbonate, polyethylene, polypropylene orpolytetrafluyoroethylene material. In one embodiment, a metal membranematerial is a chrome, gold, copper or silver material. In oneembodiment, a ceramic membrane material includes glass fiber.

There are several important advantages to the embodiments describedabove, explained in further detail below.

Breaking surface tension or capillary forces:

There are two problems caused by surface tension or capillary forceswhen pulling a liquid sample through the membrane. First (according tothe Young-Laplace theory), aqueous liquid, when placed on the surface ofthe hydrophilic membrane will spread across the surface and enter thepores of the membrane. In one embodiment, a polycarbonate track etchedmembrane is used. The pores of this membrane are well defined, and passthrough the membrane in the shape of a cylinder. The corners of thecylinder have sharp, roughly 90 degree corners. This geometry can oftenlead to a case where liquid enters the pores until it reaches the end ofthe pore at which point a meniscus develops. A pressure differentialthat would continue to drive the fluid through the membrane will beopposed by the capillary pressure caused by the interfacial forces atthe point of the meniscus. Instances have been observed where increasingthe pressure differential to drive the fluid through the membrane isopposed so strongly that the membrane deforms before flow can beinduced.

These capillary forces can be overcome by placing a membrane contactingmaterial in contact with the bottom of the membrane. This material willchange the local geometry, disrupt the meniscus and allow fluid to flowonce more. Flow through some of the pores will cause liquid to spreadacross the bottom of the membrane and contact neighboring blocked pores,disrupting their meniscuses and eventually allowing flow through thewhole area of the capture membrane sample spot.

This membrane contacting material solves the problem created by thecapillary forces but can also, on occasion cause the oppositeproblem—using a membrane contacting material can cause liquid to passthrough the membrane even without applying a pressure differential. Inother words, samples can “leak” through the membrane prematurely, beforethe vacuum is applied. This unwanted flow is not controlled and causessample variation due to its sporadic nature. It is therefore desirous inone embodiment to only apply the contacting material at the moment whenthe vacuum pressure differential is applied so that the fluid flow iscontrolled and happens when intended.

The contacting rings contacts the membrane within the samples spot whenvacuum is applied, disrupts the surface tension and allows the sample toflow through the membrane. For avoiding premature sample flow, thedesign of the manifold top is such that at rest, the membrane does nottouch the contacting rings beneath the membrane—there is a gap. However,the ring is close enough to the membrane so that that when vacuumpressure is applied, the deflection of the membrane initiates contactbetween the membrane and the contacting rings. This contact initiatesflow through the membrane.

Sample Isolation:

It is important, when conducting analytical tests, not to contaminateone sample with another. Embodiments described herein are such that manysamples can be run in parallel. It's therefore important to preventsamples within a run from intermingling and also important to preventsamples between runs from intermingling. Samples within the same run areprevented from intermingling above the membrane because they arecontained within discrete sample spots defined by the hydrophobic rings.However, underneath the membrane, samples are in contact with themanifold and, if the manifold is not designed properly, these samplescan spread across the bottom of the membrane. If the liquid does so, itcan reach another sample spot and possibly contaminate it, or block thepores from the bottom. Between runs, sample droplets that remain on themanifold can then make contact with the subsequent capture membrane thatis placed on the manifold. These droplets can contaminate the sample,discolor the membrane which is concerning to users, and they can eveninduce premature flow of the sample due to the breaking of surfacetension under the membrane.

For sample isolation during a run, the rings protrude from the surfaceand define a small contact region between the capture membrane and themanifold material itself. The rings are separated from each other anddefine a small contact region. During vacuuming, the sample may be incontact with the sample ring but will not travel along the manifold toother contact rings. In this way, each sample spot is isolated from therest. Various geometries of the hole in the manifold top can be used sothat droplets don't get trapped in the hole or, if they are trapped inthe hole they are located in a position to minimize the chance of thedroplet touching the subsequent capture membrane from the bottom.

The sample rings must have a large enough radius so that when themembrane deflects, it does not warp against a small, “sharp” ring. Tomaximize utilization of the sample spot area, the rings are located suchthat they make contact within the region of the sample spot. However,the contact region should not take up too much of the sample spot.Contacted regions will block the membrane pores and either reduce orprevent flow. This reduces the usable area of the membrane. In oneembodiment, part of the ring contact ring resides outside the perimeterof the sample spot.

Drying the Back of the Membrane for Imaging:

After samples are processed through the capture membrane it is imaged.The membrane image can be dramatically affected by droplets of waterclinging to the bottom of the membrane. This adversely affects theanalytical technique. Furthermore, liquid droplets that remain on thebottom of the membrane may also into the analytical instrument or causecross-contamination upon user handling. These droplets should be removedefficiently.

To solve this problem, a drying cassette as disclosed above wasdesigned. This cassette is used just after a sample has been processedthrough the capture membrane using the vacuum manifold. The dryingcassette has a recessed pocket to hold a capture membrane. It also has amechanically defined region that holds a blotting paper positioned to bein contact with the bottom of the capture membrane. Beneath thisblotting paper, there are 96 holes in the drying cassette that arealigned with the sample spots on the capture membrane. Furthermore, thedrying cassette is designed to nest on top of the vacuum manifold top.When the whole system is assembled (from top to bottom: the capturemembrane, the blotting paper, the drying cassette and the manifold top),a small pressure differential is generated which pushes the capturemembrane against the blotting paper and causes a small air flow. Boththe induced contact and the air flow causes efficient drying of thebottom of the membrane. The system is simple to operate and the blottingpaper, which is cut to match the proper pocket in the drying cassette,can simply be discarded.

Avoiding Loud Noises:

Air travelling between a deformable membrane and another surface cangenerate loud noises that are unpleasant and disruptive. The nature ofthe seal between the capture membrane and the contacting ring is suchthat these loud noises can be generated. These loud noises should bereduced or eliminated if possible.

To address this issue, a pressure control hole is implemented into themanifold top. This hole generates a cavity of lower pressure in thespace underneath the contacting membrane but outside the contactingrings. The hole therefore adds another zone of pressure to the system.With reference back to FIGS. 2A and 2B, the three distinct pressurezones are Zone 1: high pressure (atmospheric pressure above the plate);Zone 2: low pressure underneath the sample spot; and Zone 3: mediumpressure between the manifold top and the region of the capture membraneoutside the contacting ring. This medium zone of pressure aids the sealbetween the capture membrane and also reduces unwanted, sound-generatingair flow between the membrane and the manifold.

Sticky Residue:

When samples are processed, they can dry quickly between the capturemembrane and the manifold. Depending on the contents of the sample, asticky residue may be left behind and cause a bond between the membraneand manifold. Such a bond can rip the membrane when it is removed fromthe manifold.

To solve this issue, a variety of conventional non-stick coatings can beapplied to the manifold to reduce or prevent this effect.

EXPERIMENTAL EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseExamples, but rather should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the present invention andpractice the claimed methods. The following working examples therefore,specifically point out the preferred embodiments of the presentinvention, and are not to be construed as limiting in any way theremainder of the disclosure.

With reference now to FIG. 4, a data visualization tool 200 can be usedto help ensure that particles are being nicely dispersed on themembrane. In one embodiment, the data visualization tool 300 shows anactual image of the particles and the capture membrane (left), next to agraphical depiction of every particle (right) represented by a littleball graphic. Properties of the particle such as size andcharacteristics can be represented by the size, color and/or pattern ofthe ball graphic. Advantageously, this data visualization tool 300allows the user to verify that particles are nicely spaced out acrossthe membrane, indicative of the reliability of the data that will beobtained.

With reference now to FIG. 5, a method 400 for filtration microscopyincludes the steps of imaging the membrane 402; providing a vacuummanifold including a manifold top having a plurality of openings, and acapture membrane positioned above and spaced apart from the manifold top404; applying sample drops to sample spots on the membrane, the samplespots positioned above the plurality of openings 406; applying anegative pressure to the openings such that the capture membranedeflects and contacts a surface of the manifold top and fluid from theplurality of sample drops is suctioned through the capture membrane 408;and optically imaging particulates on the capture membrane 410. In oneembodiment, the imaging device is configured to image the filter bothbefore and after the at least one particle is captured on the filter.See for example U.S. Pat. Pub. 2017/0242234 to Ashcroft et al.,incorporated herein by reference. In one embodiment, the imaging deviceis a camera. In one embodiment, the step of optically imaging includesmatching an imaging device field of view to the size and shape of asingle hydrophobic ring for whole well imaging. In one embodiment, theplate bottom is dried after the step of applying a negative pressure. Inone embodiment, the plurality of sample drops includes abiopharmaceutical solution. In one embodiment, the biopharmaceuticalsolution includes visible particles, subvisible particles and nanometerscale particles. In one embodiment, the particulates include proteinaggregates. In one embodiment, the particulates include at least one ofpolysorbate particles, metallic particles, rubber particles, siliconeoil droplets, salt crystals, cellulose and sucrose particles. In oneembodiment, the sample drops include a viscosity between 1 cP and 200cP. Sample drop viscosity can be higher in certain embodiments. Forexample, samples of foods and oils may in certain embodiments have aviscosity of 300 cP, 500 cP, 1,000 cP, 5,000 cP, 17,000 cP or more. Inone embodiment, the sample drops include a tag to perform at least oneof fluorescent identification and protein activity monitoring. In oneembodiment, the method includes the step of applying a drop comprising atag after the step of applying a negative pressure to perform at leastone of fluorescent identification and protein activity monitoring. Inone embodiment, the method includes the step of generating a datavisualization image based on the imaging and indicative of dispersion ofthe particulates.

Now, with reference to FIGS. 6A-6K, a laminar flow fluid mechanicssimulation is shown. Advantageously, according to embodiments of thesystem described herein, as one or more pores gets blocked by a particleresting on the capture membrane (the bottom particle), the streamlinesare diverted into the next open pore or pores, and therefore stacking ofsubsequent moving particles (the top particle) on top of restingparticles is inherently engineered out of the system (see progressionshown in FIGS. 6A-6J). In the magnified view of FIG. 6K, the streamlinesare shown in more detail, diverting the top moving particle away fromthe bottom resting particle, therefore avoiding particle stacking on thecapture membrane. The laminar flow form of the Navier-Stokes equationswere solved for the domain shown in FIGS. 6A-6J and at each time-stepthe effect of the fluid mechanical forces on the particle were computedto estimate the new position. Computations were performed using acommercial finite element package. Accordingly, embodiments of thefiltration manifold assembly described herein ensures laminar flow toreduce particle stacking and produce better quality particle analysis.The vacuum manifold enables highly uniform spatial distribution ofparticles present in liquid dispersions onto a capture membrane forimproved particle analysis. Particles from the liquid sample aredeposited on the capture membrane in such a way as to minimize particleoverlap. This avoids the issue where particles that are touching oroverlapping with each other could be counted as one, larger particle,rather than two smaller ones. By optimizing laminar flow, fluid willflow around resting particles, directing other particles away from them,and particles are less likely to touch each other.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention.

What is claimed is:
 1. A vacuum manifold for filtration microscopycomprising: a manifold top comprising a plurality of protrusions eachdefining a perimeter of a plurality of openings; and a capture membranepositioned above and spaced apart from the manifold top, wherein thecapture membrane is configured to deflect into contact with theplurality of protrusions when a negative pressure is applied to theplurality of openings.
 2. The vacuum manifold of claim 1, wherein eachof the plurality of protrusions is a contacting ring.
 3. The vacuummanifold of claim 2, wherein the capture membrane further comprises aplurality of hydrophobic rings defining a plurality of sample spots. 4.The vacuum manifold of claim 3, wherein the hydrophobic rings arepositioned directly above the contacting rings.
 5. The vacuum manifoldof claim 4, wherein at least a portion of the contacting rings arepositioned directly below the sample spots.
 6. The vacuum manifold ofclaim 3 wherein at least one of the hydrophobic rings and the contactingrings are non-circular rings.
 7. The vacuum manifold of claim 1 furthercomprising: a housing defining a cavity and configured to seat themanifold top, wherein the cavity is in fluid communication with theplurality of openings.
 8. The vacuum manifold of claim 7 furthercomprising: a vacuum port in fluid communication with the cavity andconfigured to connect to a vacuum supply.
 9. The vacuum manifold ofclaim 1 further comprising: a drying cassette configured to seatblotting paper and the capture membrane.
 10. The vacuum manifold ofclaim 1, wherein the capture membrane comprises pore sizes between about50 nanometers and 10 microns.
 11. The vacuum manifold of claim 1 furthercomprising a 96 well filter plate in ANSI format.
 12. The vacuummanifold of claim 1 further comprising at least one of a 25 mm or a 47mm membrane filter.
 13. The vacuum manifold of claim 1, wherein at leasta portion of a surface of the manifold top or capture membrane comprisesa non-stick coating.
 14. The vacuum manifold of claim 1 furthercomprising: a pressure control opening configured in the manifold topand separate from the plurality of openings, wherein the pressurecontrol opening is recessed below a top surface of the plurality ofprotrusions.
 15. The vacuum manifold of claim 1, wherein a space definedby two or more of the plurality of rings, the capture membrane and themanifold top is configured to generate a pressure higher than a negativepressure applied to the plurality of openings and lower than atmosphericpressure.
 16. A method for filtration microscopy comprising: providingthe vacuum manifold of claim 1; applying a plurality of sample drops toa plurality of sample spots on the capture membrane, the plurality ofsample spots positioned above the plurality of openings; applying anegative pressure to the plurality of openings such that the capturemembrane deflects and contacts the plurality of protrusions and fluidfrom the plurality of sample drops is suctioned through the capturemembrane; and optically imaging particulates on the capture membrane.17. The method of claim 16 further comprising: optically imaging thecapture membrane prior to the step of applying a plurality of sampledrops.
 18. The method of claim 16, wherein the plurality of sample dropscomprises a biopharmaceutical solution.
 19. The method of claim 18,wherein the biopharmaceutical solution comprises visible particles,subvisible particles and nanometer scale particles.
 20. The method ofclaim 16, wherein the particulates comprise protein aggregates.
 21. Themethod of claim 16, wherein the particulates comprise at least one ofpolysorbate particles, metallic particles, rubber particles, siliconeoil droplets, salt crystals, cellulose and sucrose particles.
 22. Themethod of claim 16, wherein the sample drops comprise a viscositybetween 1 cP and 200 cP.
 23. The method of claim 16, wherein sampledrops comprise a tag to perform at least one of fluorescentidentification and protein activity monitoring.
 24. The method of claim16 further comprising: applying a drop comprising a tag after the stepof applying a negative pressure to perform at least one of fluorescentidentification and protein activity monitoring.
 25. The method of claim16 further comprising: generating a data visualization image based onthe imaging and indicative of dispersion of the particulates.
 26. Themethod of claim 16, wherein the capture membrane further comprises aplurality of hydrophobic rings defining the plurality of sample spots,wherein the step of optically imaging further comprises: matching animaging device field of view to the size and shape of a singlehydrophobic ring for whole well imaging.